The investigators are using an integrative and multidisciplinary approach to determine how the myosin heavy chain (MHC) protein drives muscle function. Myosin is the molecular motor of muscle and the major component of myofibrillar thick filaments. Its ATP-dependent interaction with actin-containing thin filaments powers muscle contraction. They will test a series of hypotheses that predict myosin properties encoded by alternative exons, and how these properties dictate the different mechanical functions of various muscles. They use the model organism Drosophila melanogaster because it has a single gene coding for muscle MHC, but produces multiple forms of the protein (isoforms) by alternative RNA splicing. Using MHC null mutants in conjunction with germline transformation, they created a series of "isoform-switch" organisms that accumulate versions of MHC differing in single domains. To determine how each alternative structural domain defines the biochemical and biophysical properties of myosin and the ultrastructural and physiological properties of muscle, they are employing a battery of in vitro and in vivo assays: ATPase, actin and nucleotide binding, in vitro motility, optical trapping, electron microscopy, whole organism muscle function and isolated fiber mechanics. As appropriate, they will create a second series of chimeric constructs, to more specifically link functional properties with structural subdomains within each alternative region of the myosin head. Defining whether in vitro properties dictate in vivo functions is difficult, since a biochemical activity of a protein may always correlate with a particular mechanical property of a muscle without there being a causal relationship. The Drosophila muscle system is unique in that the effects of individual functional domains can be tested in muscle cells and intact organisms. Therefore, they can determine directly and to what degree a specific biochemical property defines a mechanical characteristic. They will also use these assays to test hypotheses regarding the molecular, biochemical, physiological and ultrastructural defects associated with two Mhc mutations that affect key amino acid residues. Their results will be interpreted in relation to the three-dimensional structure of the myosin molecule and models for the mechanochemical cycle. Overall, their novel approach will yield direct insight into how the myosin protein functions in muscle and permit testing of models for the transduction of chemical energy into movement. Since mutations in the myosin head cause defects in human cardiac and skeletal muscle, these studies are relevant to understanding human myopathies.